Inhibitors of DP-mediated processes, compositions, and therapeutic methods thereof

ABSTRACT

Compounds selected from those of general formula                    
     and A is selected from specified aminoacyl compounds are inhibitors of DP-IV mediated processes.

This application is a divisional of application Ser. No. 08/647,887,filed Aug. 27, 1996 now U.S. Pat. No. 5,939,560, which is a nationalstage of PCT/GB94/0265, filed Nov. 30. 1994.

BACKGROUND

DP-IV (EC 3.4.14.5) is a membrane-bound serine protease first identifiedin rat kidney by its ability to cleave dipeptides from the N-terminus ofcertain peptides (Hopsu-Havu, V. K. and Glenner, G. G., Histochemie,1966, 2, 197). The dipeptides must be of the type X-Pro or X-Ala whereX=any amino acid X-Proline is more efficiently cleaved than X-Ala.

DP-IV is widely distributed in mammalian tissues and is found in greatabundance in the kidney, intestinal epithelium and placenta (Yaron, A.and Naider, F., Critical Reviews in Biochem. Mol. Biol. 1993, 28 (1),31). In the human immune system the enzyme is expressed almostexclusively by activated T-lymphocytes of the CD4⁺ type where the enzymehas been shown to be synonymous with the cell-surface antigen CD26.

The exact role of DP-IV in human physiology is not completely understoodbut recent research has shown that the enzyme clearly has a major rolein human physiology and pathophysiology, eg.

(a) The immune response: DP-IV expression is increased in T-cells uponmitogenic or antigenic stimulation (Mattem, T. et al., Scand. J.Immunol. 1991, 33, 737). It has been reported that inhibitors of DP-IYand antibodies to DP-IV suppress the proliferation of mitogen- andantigen-stimulated T-cells in a dose-dependant manner (Schön, E. et al.,Biol. Chem. Hoppe-Seyler, 1991, 372, 305 and refs. within).

 Various other functions of T-lymphocytes such as cytokine production,IL-2 mediated cell proliferation and B-cell helper activity have beenshown to be dependant on DP-IV activity (Schoin, E. et al., Scand. J.Immunol. 1989, 29, 127). Recently, DP-W inhibitors based on boroprolinewhere reported (Flentke, G. R. et al., Proc. Nat. Acad. Sci. USA, 1991,88, 1556) which, although unstable, were effective in inhibitingantigen-induced lymphocyte proliferation and IL-2 production in murineCD4⁺ T-helper cells. Such boronic acid inhibitors have been shown tohave an effect in vivo in mice causing suppression of antibodyproduction induced by immune challenge (Kubota, T. et al., Clin. Exp.Immunol. 1992, 89 192). Other recent papers also provide evidence forthe involvement of DP-IV in the immune response (eg. Tanaka, T. et al.,Proc. Natl. Acad. Sci. N.Y., 1993, 9, 4586; Hegen, M. et al., CellImmun. 1993, 146, 249; Subramanyan, M. et al., J. Immunol. 1993, 150,2544).

 The importance of DP-IV is attributed by some investigators to itscell-surface association with the transmembrane phosphatase CD45(Torimoto, Y. et al., J. Immunol. 1991, 147, 2514). The CD45 - DP-IVassociation is possibly disrupted by DP-IV inhibitors or non-active siteligands. CD45 is known to be an integral component of T-cell signaling.

(b) Recently, a press release from the Pasteur Institute in Paris (andsubsequently a presentation by A. G. Hovanessian at the 8th Cent. GardesMeeting, Paris, 25-27th October 1993) reported that DP-IV was essentialfor the penetration and infectivity of HIV-1 and HIV-2 viruses in CD4⁺T-cells. The French group claimed that DP-IV interacted with and mayhave cleaved the V3 loop of the gp120 envelope glyco-protein of thevirus. They also reported that inhibitors or antibodies to DP-IVsuccessfully prevented entry of the virus into cells. It was knownpreviously that there is a selective decrease of CD26 expression inT-cells from HIV-1 infected individuals (Valle-Blazquez, M et al., J.Immunol. 1992, 149, 3073), and that HIV-1 Tat protein binds to DP-IV(Subramanyam, M. et al., J. Immunol. 1993, 150, 2544).

(c) It has been shown recently that lung endothelial DP-IV is anadhesion molecule for lung-metastatic rat breast and prostate carcinomacells (Johnson, R. C. et al., J. Cell. Biol. 1993, 121, 1423). DP-IV isknown to bind to fibronectin and some metastatic tumour cells are knownto carry large amounts of fibronectin on their surface.

(d) DP-IV has been shown to associate with the enzyme adenosinedeaminase (ADA) on the surface of T-cells (Kameoka, J. et al., Science,193, 26 466). ADA deficiency causes severe combined immunodeficiencydisease (SCID) in humans This ADA-CD26 interaction may provide clues tothe pathophysiology of SCID.

(e) High levels of DP-IV expression have been found in human skinfibroblast cells from patients with psoriasis, rheumatoid arthritis (RA)and lichen planus (Raynaud, F. et al., J. Cell. Physiol. 1992, 151,378).

(f) High DP-IV activity has been found in tissue homogenates frompatients with benign prostate hypertrophy and in prostatosomes. Theseare prostate derived organelles important for the enhancement of spermforward motility (Vanhoof, G. et al., Eur. J. Clin. Chem. Clin. Biochem.1992, 30, 333).

(g) DP-IV has been shown to be responsible for the degradation andinactivation of circulating peptides with penultimate proline or alanineat the N-terminus, eg. substance P, growth hormone releasing factor andmembers of the glucagon/vasoactive intestinal peptide family (Menthein,R. et al., Eur. J. Biochem. 1993, 214, 829).

(h) Raised levels of DP-IV have been observed in the gingiva of patentswith periodontitis (Cox, S. W. et al., Arch. Oral. Biol. 1992, 37, 167).

(i) There are also a number of other reports of raised (or sometimeslowered) levels of DP-IV in various pathological conditions.

It follows from the above that potent inhibitors of DP-IV may be usefulas drugs for the treatment of human disease. Such inhibitors could beuseful as:

(a) Immunosuppressants, eg. in organ transplantation; cytoline releasesuppressants eg. in various autoimmune diseases such as inflammatorybowel disease, multiple sclerosis, RA.

(b) Drugs for the prevention of HIV entry into T-cells and thereforeuseful in the prophylaxis and treatment of AIDS.

(c) Drugs for the prevention of metastases, particularly of breast andprostate tumours to the lungs.

(d) Agents to treat dermatological diseases, eg. psoriasis, lichenplanus.

(e) Drugs to suppress sperm motility and therefore act as malecontraceptive agents.

(f) Agents beneficial in benign prostate hypertrophy.

Inhibitors of DP-IV

The only competitive inhibitors of DP-IV enzyme activity reported so farare the unstable boronic acids (t½ 30-90 min at pH 7) mentioned above.(Bachovchin et al, WO 91/16339, October 1991) having K_(i) values in thenanomolar range for DP-IV, and simple amino-acid pyrrolidides orthiazolides (Neubert et al., DD 296 075 A5, November 1991) which haveonly modest potency (K_(i)>0.1 μM). Amino-acyl proline aldehydes claimedin the same German patent cannot be synthesised due to a facileintramolecular condensation of the N-terminal amino group with thealdehyde function.

We now disclose highly potent competitive inhibitors of DP-IV (withK_(i) values in the 10⁻⁶−10⁻¹⁰ range) which are also chemically stable(t½>24 h). They fall into three broad groups of compounds (Groups I, IIand III).

GROUP I

These are molecules designed to bind tightly in the active site of DP-IYand to inhibit its proteolytic activity without interfering withattachment of any accessory ligands which may bind to the surface ofDP-IV (i.e. not at its active site). Such Group I compounds could beuseful as immunosuppressants; anti-HIV infectivity agents; agents tosuppress release of certain cytokines (eg. IL-2, IL-6, γ-INF) fromactivated T-cells. The boronic acids and pyrolidides referred to earlieralso fall into this category.

GROUP II

These are evolved from Group I compounds; however they containlong-chain extensions to the side-chains of the amino-acid defined as Ain the general structure. The resulting compounds bind tightly to theactive-site of DP-IV but the long-chain extensions protrude from theenzyme active site and serve to prevent the attachment of any otherligand which may bind to the surface of DP-IV. Such compounds could havethe same uses as Group I compounds but in addition could block theinteraction of DP-IY with (i) CD45 (ii) the gp 120 V3 loop of HIV-1(iii) tumour cell surface fibronectin (iv) any other ligand importantfor T-cell activation, virus entry into T-cells or tumour cell adhesion.

GROUP III

This group comprises novel dimers in which two active-site directedinhibitors of DP-IV are linked via the side-chains of their amino-acidresidues designated A in the general structure by a long chain. Suchdimers can inhibit two molecules of DP-IV concurrently and also preventaccessory ligands binding to the surface of DP-IV. These dimers wouldhave the same uses as Group II compounds but may be more effective.

The invention provides inhibitors of DP-IV mediated processes, theinhibitors being of general formula:

A is attached to Y;

—Y=—N, —CH or ═C (when the —CO group of A is replaced with CH═ or CF═);

R=H, CN, CHO, B(OH)₂, C≡C—R₇, or CH═N—R₈;

R₇=H, F, lower alkyl (C₁ to C₆), CN, NO₂, OR₉, CO₂R₉ or COR₉;

R₈=Ph, OH, OR₉, OCOR₉, or OBn;

R₉=lower alkyl (C₁-C₆); and either ω or both ε's may be absent.

The structure of A is dependent on the nature of R in moiety B and onthe nature of the group to which the resulting compound belongs.

Group I Compounds

(a) R=H

A is an α-amino-acyl group derived from an α-amino-acid bearing acycloaliphatic side-chain (e.g. C₄ to C₁₀, mono or bicyclic) whose ringmay contain one or more heteroatoms e.g. L-cyclohexylglycine,L-cyclopentylglycine, L-decahydronaphthylglycine, L-piperidylglycine;

 or

A is a β-amino-acyl group of general formula

 where p=1-6 and the ring may also contain one or more heteroatomsreplacing CH₂ unit(s).

Both α and β-amino acyl groups in (a) above may contain unsaturation intheir rings e.g.

 and also may contain one or more heteroatoms.

(b) R=CN; C≡C—R₇ or CH═N—R₈

A is as defined in (a) above but in addition may be derived from anyL-α-amino acid bearing a lipophilic side-chain, eg. Ile.

(c) R=CHO or B(OH)₂

A is a β-amino-acyl group as defined in (a) above. The resulting A-Bcompounds are stable, unlike α-aminoacyl derivatives of the same typewhich undergo a facile intramolecular cyclisation. In compounds (c)B(OH)₂ may be present as a boronate ester eg.

 these being labile in water giving the free boronic acids.

Group II Compounds

Where R═H, CN, C≡C—R₇ or CH═N—R₈, A is an α-amino acid derivative whoseside-chain carries a functional group which is derivatised to produce along chain terminating in various groups R₃. A may be of the followingthree types of structure:

where

 a=1-5; D=G—(CH₂)_(b)(R₄)_(q)—R₃; G=O, NH, or NMe;

b=0-12; q=0-5;

D¹=D with G≠O;

R₄=Z—NH—(CH₂)_(c)— or NH—Z—(CH₂)_(c)— where c=1-12 and Z=CO, CH₂ or SO₂;and

R₃=CO₂H or ester [e.g. any lower alkyl, fluoroalkyl or cycloalkyl (C₁ toC₈), or aromatic or heteroaromatic (5 or 6membered rings, mono- orbicylic) ester] thereof; CONH₂; CONHNH₂; CONR₅R₆; CONHNR₅R₆; PO₃H (orester thereof e.g. as defined under CO₂H); SO₃H; SO₂NH₂; SO₂NR₅R₆; OH;OR₅; aryl or heteroaryl (e.g. 5 or 6membered rings, monocyclic orbicyclic) [including substituted aryl or heteroaryl with substituentspreferably chosen from F, Cl, I, Br, OH, OR₅, NO₂, SO₃H, SO₂NH₂,SO₂NR₅R₆, NH₂, NR₅R₆, CO₂R₅, CF₃, CN, CONH₂, CONR₅R₆, NHCO₂R₅,CH(:NR₅)NR₅R₆, NH—CH(:NR₅)NR₅R₆ and R₅]; NH₂; NR₅R₆; NHCO₂R₅;NHSO₂NR₅R₆; NHCOR₅; NH—SO₂R₅; NH—CH(:NR₅)NR₅R₆; NHCONR₅R₆; sugar (whichmay be attached via an ether or a glycosidic bond); CO-aminosugar(attached via the —NH₂) eg. glucosamine or galactosamine;NHCO-aminosugar; or NHCS-aminosugar.

 In the above definition of R₃ “sugar” refers to any carbohydrate oroligosaccharide, and R₅ and R₆ are independently selected from H andalkyl, fluoroallyl and cycloalkyl groups (of up to 8 atoms), aryl,heteroaryl and alkylheteroaryl groups (of up to 11 atoms) or R₅ and R₆together comprise a chain and (C₃ to C₈).

where R¹=H, Me; the ring may also contain more heteroatoms;

E=J—(CH₂)_(b)—(R₄)_(q)—R₃; J=CO, CH₂ or SO₂; and a, b, q, R₃ and R₄ asdefined under (i)

where R²=H or Me; the ring may also contain one or more heteroatoms;

L=(CH₂)_(d)—[CO]_(r)—(CH₂)_(b)—(R₄)₁—R₃ or(CH₂)_(e)—NR¹—(CH₂)_(b)—(R₄)_(q)—R₃; r=0 or 1; d=0-4; e=2-4; and b, q,R₃ and R₄ as defined under (i).

Group III

Group III compounds are defined by the general formula:

where ω=CH₂, O, NH, CO, S, SO₂, Ph or NMe and, independently, ε=CH₂, O,NH, CO, S, SO₂, Ph or NMe.

These compounds are symmetrical dimers. They may have any B structure asdefined previously. A may be chosen from any group II structure [(i),(ii) or (iii)], but in this case he terminal group R₃ in each A residueis deleted and replaced with a shared symmetrical group [ε—ωε] whichconnects the two halves of the dimer; ω may be absent, in which caseboth ε's are joined together to constitute the chain linking the two A-Bmoieties; alternatively both ε's may be absent in which case to solelyjoins the two A-B moieties.

The stricture of ε—ω—ε must of course be chemically feasible eg.NH—CO—NH, CO—NH—CO—, SO₂—NMe—SO₂; it will be obvious to those skilled inthe art which structures are not feasible, eg. —NH—NH—NH—. A specificpossible example is shown in Table 7.

In such compounds as described under Groups II and III certain —CH₂—groups present in the long chains could be replaced with knownbioisosteres eg. —O— without affecting inhibitory or binding activitytowards DP-IV. Also such groupings as —CONHCH₂CH₂NHCO if they occurcould be replaced by eg.

Further, for compounds in Groups I, II and III any amide bond connectingA and B or any amide in the side-chains of A (in Groups II and E) may bereplaced by known bioisosteres of amides eg.

See Table 8 for examples of such replacements.

Biochemistry

All compounds were tested in vitro against pure human DP-IV (purchasedfrom M & E, Copenhagen, Denmark). Inhibition of DP-IV was determinedusing the fluorescent substrate Ala—Pro—AFC (K_(m)0.8 μM) at threeconcentrations for each inhibitor. A typical assay (total volume 0.4 ml)comprised sodium Hepes 83.3 mM, EDTA 1.67 mM, BSA 1.5 mg ml⁻¹ pH 7.8,DP-IV 25 μU ml⁻¹, inhibitor (in 10 mM acetate pH 4.0). The reaction wasstarted by the addition of substrate and readings taken every 30 s for7.5 min, excitation at 395 nm, emission 450 nm. K_(i) values weredetermined using Dixon plots.

Chemistry

152 Examples of compounds synthesised are shown in Tables 1-8 followedby schemes and experimental details for the preparation of differentstructural types. All final products were characterised by FAB massspectrometry and purity assessed by reverse phase hplc; allintermediates were characterised by ¹H NMR.

Table 9 shows selected K_(i) values against DP—IV determined forinhibitors of different structural types.

TABLE 1 Examples of Group I (a) Calculated FAB Mass No. A X R n FormulaMol. Wt. spec. [M + H]⁺ 1

CH₂ H 1 C₁₁H₂₀N₂O 196.2 197.2 2

CH₂ H 1 C₁₂H₂₂N₂O 210.2 211.2 3

CH₂ H 1 C₁₀H₂₀N₂O 184.2 185.2 4

CH₂ H 1 C₁₂H₂₀N₂O 208.2 209.2 5 cis

CH₂ H 1 C₁₁H₂₀N₂O 196.1 197.2 6 trans

CH₂ H 1 C₁₁H₂₀N₂O 196.1 197.2 7 trans

CH₂ H 1 C₁₁H₁₈N₂O 194.1 195.2 8 trans

CH₂ H 1 C₁₀H₁₈N₂O 182.1 183.2 9

CH₂ H 1 C₁₁H₁₄N₂O 190.1 191.2 10 trans

CH₂ H 1 C₁₃H₂₄N₂O 224.2 225.2

TABLE 2 Examples of Group I (b)

Calculated FAB Mass No. A X n R¹ R Formula Mol. Wt. spec. [M + H]⁺ 11H-Ile CH₂ 1 H CN C₁₁H₁₉N₃O 209.3 210.2 12 H-Lys(Z) CH₂ 1 H CN C₁₉H₂₆N₄O₃358.2 359.2 13 H-Pro CH₂ 1 H CN C₁₀H₁₅N₃O 193.1 194.1 14

CH₂ 1 H CN C₉H₁₃N₃OS 211.1 212.2 15

CH₂ 1 H CN C₉H₁₃N₃OS 211.1 212.2 16

CH₂ 1 H CN C₁₃H₂₁N₃O 235.2 236.3 17

CH₂ 1 H CN C₁₂H₁₉N₃O 221.2 222.2 18

CH₂ 1 H CN C₁₁H₁₉N₃O 209.2 210.2 19 H-Ile S 1 H CN C₁₀H₁₇N₃OS 227.1228.1 20 H-Ile S 1 CN H C₁₀H₁₇N₃OS 227.1 228.1 21

S 1 H CN C₁₂H₁₉N₃OS 253.1 254.1 22 H-Lys(Z) S 1 H CN C₁₈H₂₄N₄O₃S 376.23.2 23

S 1 H CN C₁₁H₁₇N₃OS 239.1 240.2 24 H-Ile O 1 H CN C₁₀H₁₇N₃O₂ 211.1 212.225 H-Ile CH₂ 2 H CN C₁₂H₂₁N₃O 223.2 224.2 26 H-Ile S 2 H CN C₁₁H₁₉N₃OS241.1 242.1 27 H-Ile SO₂ 1 H CN C₁₀H₁₇N₃O₃S 259.1 260.1 28 H-Ile

1 H CN C₁₀H₁₇N₃O₂S 243.1 244.1 29 H-Ile

1 H CN C₁₀H₁₇N₃O₂S 243.1 244.2 30

CH₂ 1 H CN C₁₂H₁₉N₃O 221.2 222.2 31

CH₂ 1 H CN C₁₂H₁₉N₃O 221.2 222.2 32

CH₂ 1 H CN C₁₁H₁₇N₃O 207.2 208.2 33

CH₂ 1 H CN C₁₁H₁₇N₃O 207.2 208.2 34

CH₂ 1 H CN C₁₂H₁₇N₃O 219.1 220.1 35

CH₂ 1 H CN C₁₂H₁₇N₃O 219.1 220.1 36

CH₂ 1 H CN C₁₂H₁₉N₃O 221.2 222.2 37

CH₂ 1 H CN C₁₂H₁₇N₃O 219.1 220.1

TABLE 3 Example of Group I (c)

Calculated FAB Mass No. A X R n Formula Mol. Wt. spec. [M + H]⁺ 38

CH₂ CHO 1 C₁₂H₂₀N₂O₂ 224.2 225.2 39

CH₂ CHO 1 C₁₁H₁₈N₂O₂ 210.2 211.2 40

CH₂ CHO 1 C₁₁H₁₈N₂O₂ 210.2 211.2 41

CH₂ B* 1 C₂₀H₃₃BN₂O₃ 360.3 361.3 42

CH₂ B* 1 C₂₁H₃₅BN₂O₃ 374.3 375.1 43

CH₂ B* 1 C₂₁H₃₅BN₂O₃ 374.3 375.1 44

CH₂ B* 1 C₂₁H₃₃BN₂O₃ 372.3 373.3 45

CH₂ B* 1 C₂₁H₃₃BN₂O₃ 372.3 373.3 B*=

TABLE 4 Examples of Group II (i)

Calculated FAB Mass No. n Q X m R Formula Mol. Wt. spec. [M + H]⁺ 46 1—CONHCH₂CO₂Bn CH₂ 1 H C₁₇H₂₃N₃O₄ 333.2 334.2 47 1 —CONHCH₂CO₂H CH₂ 1 HC₁₀H₁₇N₃O₄ 243.1 244.2 48 1 —CONH(CH₂)₃CO₂H CH₂ 1 H C₁₂H₂₁N₃O₄ 271.2272.2 49 1 —CONH(CH₂)₂CO₂Bn CH₂ 1 H C₁₈H₂₅N₃O₄ 347.2 348.2 50 1—CONH(CH₂)₂CO₂H CH₂ 1 H C₁₁H₁₉N₃O₄ 257.1 258.2 51 1 —CONH(CH₂)₅CO₂Bn CH₂1 H C₂₁H₃₁N₃O₄ 389.3 390.3 52 1 —CONH(CH₂)₅CO₂H CH₂ 1 H C₁₄H₂₅N₃O₄ 299.2300.2 53 1 —CONH(CH₂)₃CO₂Bn CH₂ 1 H C₁₉H₂₇N₃O₄ 361.2 362.2 54 2—CONHCH₂CO₂Bn CH₂ 1 H C₁₈H₂₅N₃O₄ 347.2 348.2 55 2 —CONHCH₂CO₂H CH₂ 1 HC₁₁H₁₉N₃O₄ 257.1 258.1 56 2 —CONH(CH₂)₂CO₂Bn CH₂ 1 H C₁₉H₂₇N₃O₄ 361.2362.3 57 2 —CONH(CH₂)₃CO₂Bn CH₂ 1 H C₂₀H₂₉N₃O₄ 375.2 376.3 58 2—CONH(CH₂)₃CO₂H CH₂ 1 H C₁₃H₂₃N₃O₄ 285.2 286.2 59 2 —CONH(CH₂)₅CO₂Bn CH₂1 H C₂₂H₃₃N₃O₄ 403.3 404.3 60 2 —CONH(CH₂)₅CO₂H CH₂ 1 H C₁₅H₂₇N₃O₄ 313.2314.2 61 2 —CONH(CH₂)₂CO₂H CH₂ 1 H C₁₂H₂₁N₃O₄ 271.2 272.2 62 2—CONH(CH₂)₇CO₂Bn CH₂ 1 H C₂₄H₃₇N₃O₄ 432.3 432.4 63 2 —CONH(CH₂)₇CO₂H CH₂1 H C₁₇H₃₁N₃O₄ 341.3 342.5 64 2 —CONH(CH₂)₇CONH—(CH₂)₃NHZ CH₂ 1 HC₂₈H₄₅N₅O₅ 531.3 532.3 65 2 —CONH(CH₂)₆CONH—(CH₂)₅CO₂Bn CH₂ 1 HC₂₉H₄₆N₄O₅ 530.4 531.2 66 2 —CONH(CH₂ ₆CONH—(CH₂)₅CO₂H CH₂ 1 HC₂₂H₄₀N₄O₅ 440.3 441.3 67 2 —CONH(CH₂)₇CONH—(CH₂)₃NH₂ CH₂ 1 H C₂₀H₃₉N₅O₃397.3 398.3 68 2 —CONH(CH₂)₁₁CO₂Bn CH₂ 1 H C₂₈H₄₅N₃O₄ 487.3 488.4 69 2—CONH(CH₂)₁₁CO₂H CH₂ 1 H C₂₁H₃₉N₃O₄ 397.3 398.3 70 2 —CONH(CH₂)₆CO₂BnCH₂ 1 H C₂₃H₃₅N₃O₄ 417.3 418.3 71 2 —CONH(CH₂)₆CO₂H CH₂ 1 H C₁₆H₂₉N₃O₄327.2 328.2 72 2 —CONH(CH₂)₅CONH—CH₂CF₃ CH₂ 1 H C₁₇H₂₉F₃N₄O₃ 394.2 395.373 2 —CONH(CH₂)₅CONH—CH₂(CF₂)₂CF₃ CH₂ 1 H C₁₉H₂₉F₇N₄O₃ 494.2 495.2 74 2—CONH(CH₂)₅CONH—(CH₂)₆OH CH₂ 1 H C₂₁H₄₀N₄O₄ 412.3 413.2 75 2—CONH(CH₂)₅CONH—(CH₂)₃Ph CH₂ 1 H C₂₄H₃₈N₄O₃ 430.3 431.2 76 2—CONH(CH₂)₅CONH—(CH₂)₄Ph CH₂ 1 H C₂₅H₄₀N₄O₃ 444.3 445.2 77 2—CONH(CH₂)₅CON-(^(n)Bu)₂ CH₂ 1 H C₂₃H₄₄N₄O₃ 424.3 425.3 78 2—CONH(CH₂)₅CON-(^(n)Hx)₂ CH₂ 1 H C₂₇H₅₂N₄O₃ 480.4 481.4 79 2—CONH(CH₂)₅CONH—CH₂Ph CH₂ 1 H C₂₂H₃₄N₄O₃ 402.3 403.4 80 2—CONH(CH₂)₄CO₂Bn CH₂ 1 H C₂₁H₃₁N₃O₄ 389.2 390.3 81 2 —CONH(CH₂)₄CO₂H CH₂1 H C₁₄H₂₅N₃O₄ 299.2 300.3 82 2 —CONH(CH₂)₅CONH—CH₂CH₃ CH₂ 1 HC₁₇H₃₂N₄O₃ 340.3 341.3 83 2 —CONH(CH₂)₆OH CH₂ 1 H C₁₅H₂₉N₃O₃ 299.2 300.384 2 —CONH(CH₂)₅CO-1-Pip CH₂ 1 H C₂₀H₃₆N₄O₃ 380.3 381.4 85 2—CONH(CH₂)₅CONH₂ CH₂ 1 H C₁₅H₂₈N₄O₃ 312.2 313.3 86 2—CONH(CH₂)₅CONH—(CH₂)₉CH₃ CH₂ 1 H C₂₅H₄₈N₄O₃ 452.4 453.5 87 2—CONH(CH₂)₅CONH—(CH₂)₆CH₃ CH₂ 1 H C₂₂H₄₂N₄O₃ 410.3 411.4 88 2—CONH(CH₂)₅CONH—CH₂Ch CH₂ 1 H C₂₂H₄₀N₄O₃ 408.3 409.4 89 2—CONH(CH₂)₅CONH—(CH₂)₃NHZ CH₂ 1 H C₂₆H₄₁N₅O₅ 503.3 504.4 90 2—CONH(CH₂)₅CONH—(CH₂)₃NH₂ CH₂ 1 H C₁₈H₃₅N₅O₃ 369.3 370.3 91 2—CONH(CH₂)₅CONH—(CH₂)₃—Gua CH₂ 1 H C₁₉H₃₇N₇O₃ 411.3 412.4 92 2—CONH(CH₂)₅CONH—Ph(4-SO₃H) CH₂ 1 H C₂₁H₃₂N₄O₆S 468.2 469.2 93 2—CONH(CH₂)₅CONH-4-Pip(1-Bn) CH₂ 1 H C₂₇H₄₃N₅O₃ 485.3 486.3 94 2—CONH(CH₂)₅CONH-4-Pip CH₂ 1 H C₂₀H₃₇N₅O₃ 395.3 396.3 95 2—CONH(CH₂)₄N(Z)—(CH₂)₃NHZ CH₂ 1 H C₃₂H₄₅N₅O₆ 595.3 596.3 96 2—CONH(CH₂)₄NH—(CH₂)₃NH₂ CH₂ 1 H C₁₆H₃₃N₅O₂ 327.2 328.2 97 2—CONH(CH₂)₅CO₂Bn CH₂ 1 CN C₂₃H₃₂N₄O₄ 428.3 429.3 98 3—CONH(CH₂)₆CONH—(CH₂)₅CO₂Bn CH₂ 1 H C₃₀H₄₈N₄O₅ 544.4 545.2 99 3—CONH(CH₂)₆CONH—(CH₂)₅CO₂H CH₂ 1 H C₂₃H₄₂N₄O₅ 454.3 455.3 100  3—CONH(CH₂)₅CO₂Bn CH₂ 1 H C₂₃H₃₅N₃O₄ 417.3 418.2 101  3 —CONH(CH₂)₅CO₂HCH₂ 1 H C₁₆H₂₉N₃O₄ 327.2 328.2 102  2 —SO₂NH(CH₂)₅CO₂H CH₂ 1 HC₁₄H₂₇N₃O₅S 349.2 350.2 103  2 —CONH(CH₂)₈NH—G* CH₂ 1 H C₂₄H₄₅N₅O₇S547.4 548.5 G*=

TABLE 5 Example of Group II (ii)

Calculated FAB Mass No. n Q X m R Formula Mol. Wt. spec. [M + H]⁺ 104 1—CO(CH₂)₆CO₂H CH₂ 1 H C₁₅H₂₇N₃O₄ 313.2 314.3 105 1 —CO(CH₂)₆CO₂Bn CH₂ 1H C₂₂H₃₃N₃O₄ 403.3 404.3 106 3 —CO(CH₂)₄CO₂H CH₂ 1 H C₁₅H₂₇N₃O₄ 313.2314.3 107 3 —CO(CH₂)₄CO₂Me CH₂ 1 H C₁₆H₂₉N₃O₄ 327.2 328.3 108 4—CO(CH₂)₅NH₂ CH₂ 1 H C₁₆H₃₂N₄O₂ 312.3 313.3 109 4 —CO(CH₂)₃NH₂ CH₂ 1 HC₁₄H₂₈N₄O₂ 284.2 285.2 110 4 —CO(CH₂)₃NHSO₂Pfp CH₂ 1 H C₂₀H₂F₅N₄O₄S514.2 515.2 111 4 —CO(CH₂)₃NHCOPfp CH₂ 1 H C₂₁H₂₇F₅N₄O₃ 478.2 479.2 1124 —CO(CH₂)₃NHSO₂—CH₂CF₃ CH₂ 1 H C₁₆H₂₉F₃N₄O₄S 430.2 431.3 113 4—CO(CH₂)₁₁NHCO—(CH₂)₆NHZ CH₂ 1 H C₃₇H₆₃N₅O₅ 657.5 658.6 114 4—CO(CH₂)₁₁NH—CO(CH₂)₆NH₂ CH₂ 1 H C₂₉H₅₇N₅O₃ 523.4 524.4 115 4—CO(CH₂)₅NHCO—(CH₂)₅NHCO(CH₂)₅—NHZ CH₂ 1 H C₃₆H₆₀N₆O₆ 672.5 673.6 116 4—CO(CH₂)₅NHCO—(CH₂)₅NHCO(CH₂)₅—NH₂ CH₂ 1 H C₂₈H₅₄N₆O₄ 538.4 539.4 117 4—CO(CH₂)₃CO₂H CH₂ 1 H C₁₅H₂₇N₃O₄ 313.2 314.3 118 4 —CO(CH₂)₃CO₂Bn CH₂ 1H C₂₂H₈₃N₃O₄ 403.3 404.3 119 4 —CO(CH₂)₆NH₂ CH₂ 1 H C₁₇H₃₄N₄O₂ 326.3327.3 120 4 —CO(CH₂)₇NH₂ CH₂ 1 H C₁₈H₃₆N₄O₂ 340.3 341.3 121 4—CO(CH₂)₁₆Me CH₂ 1 H C₂₈H₅₅N₃O₂ 465.4 466.4 122 4 —CO(CH₂)₆—Gua CH₂ 1 HC₁₈H₃₆N₆O₂ 368.3 369.3 123 4 —SO₂(CH₂)₇CH₃ CH₂ 1 H C₁₈H₃₇N₃O₃S 375.3376.3 124 4 —CO(CH₂)₁₁NH₂ CH₂ 1 H C₂₂H₄₄N₄O₂ 396.4 397.4 125 4 —COCH₂NHZCH₂ 1 H C₂₀H₃₀N₄O₄ 390.2 391.3 126 4 —CO(CH₂)₂NHZ CH₂ 1 H C₂₁H₃₂N₄O₄404.2 405.3 127 4 —CO(CH₂)₃NHZ CH₂ 1 H C₂₂H₃₄N₄O₄ 418.3 419.3 128 4—CO(CH₂)₂NH₂ CH₂ 1 H C₁₂H₂₄N₄O₂ 256.2 257.2 129 4 —CO(CH₂)₅NHZ CH₂ 1 HC₂₄H₃₈N₄O₄ 446.3 447.4 130 4 —COCH₂—Gua CH₂ 1 H C₁₃H₂₆N₆O₂ 298.2 299.3131 4 —CO(CH₂)₂NH₂ CH₂ 1 H C₁₃H₂₆N₄O₂ 20.2 271.3 132 4 —CO(CH₂)₂—Gua CH₂1 H C₁₄H₂₈N₆O₂ 312.2 313.3 133 4 —CO(CH₂)₃—Gua CH₂ 1 H C₁₅H₃₀N₆O₂ 326.3327.3 134 4 —CO(CH₂)₅—Gua CH₂ 1 H C₁₇H₃₄N₆O₂ 354.3 355.3 135 4—CO(CH₂)₆NH₂ CH₂ 1 CN C₁₈H₃₃N₅O₂ 351.3 352.4 136 4 —CO(CH₂)₇NH₂ CH₂ 1 CNC₁₉H₃₅N₅O₂ 365.3 366.3

TABLE 6 Examples of Group II (iii)

Calculated FAB Mass No. R R¹ X n Y Formula Mol. Wt. spec. [M + H]⁺ 137 H—OCH₂CONH(CH₂)₅—CO₂H CH₂ 1 H C₁₅H₂₇N₃O₅ 329.2 330.3 138 H—OCH₂CONH(CH₂)₅—CO₂Bn CH₂ 1 H C₂₂H₃₃N₃O₅ 419.3 420.3 139 H—OCH₂CONH(CH₂)₄—CO₂Bn CH₂ 1 H C₂₁H₃₁N₃O₅ 405.2 406.3 140 H—OCH₂CONH(CH₂)₄—CO₂H CH₂ 1 H C₁₄H₂₅N₃O₅ 315.2 316.3 141 CH₃ —OCH₃ CH₂ 1H C₉H₁₈N₂O₂ 186.1 187.2 142 CH₃ —OC₂H₅ CH₂ 1 H C₁₀H₂₀N₂O₂ 200.1 201.2143 CH₃ —O(CH₂)₅CH₃ CH₂ 1 H C₁₄H₂₈N₂O₂ 256.2 257.3 144 CH₃—OCH₂CONH(CH₂)₅—CO₂Bn CH₂ 1 H C₂₃H₃₅N₃O₅ 433.3 434.3 145 CH₃—OCH₂CONH(CH₂)₅—CO₂H CH₂ 1 H C₁₆H₂₉N₃O₅ 343.2 344.3 146 CH₃—OCH₂CONH(CH₂)₄—CO₂Bn CH₂ 1 H C₂₂H₃₃N₃O₅ 419.2 420.3 147 CH₃—OCH₂CONH(CH₂)₄—CO₂H CH₂ 1 H C₁₅H₂₇N₃O₅ 329.2 330.3

TABLE 7 Example of Group III Calculated FAB Mass No. Structure FormulaMol. Wt. spec. [M + H]⁺ 148

C₃₂H₅₄N₈O₄ 614.4 615.4

TABLE 8 Specific examples of compounds A-B, containing amide bondbioisosteres. FAB Mass Calculated spec. No. A-B Formula Mol. Wt. [M +H]⁺ 149

C₁₁H₂₁N 167.2 168.2 150

C₁₂H₂₀N₂ 192.2 193.2 151

C₁₂H₂₀N₂ 192.2 193.2 152

C₁₀H₂₀N₂S 200.1 201.2

TABLE 9 Selected K_(i) values against DP-IV. No. K_(i)(M)  2 6.4 × 10⁻⁸ 7 7.6 × 10⁻⁶ 11 2.2 × 10⁻⁹ 20 1.7 × 10⁻⁹ 23  5.0 × 10⁻¹⁰ 35 3.7 × 10⁻⁸38 9.8 × 10⁻⁹ 44 2.0 × 10⁻⁹ 59 1.5 × 10⁻⁷ 66 1.8 × 10⁻⁷ 97  5.0 × 10⁻¹⁰110  2.5 × 10⁻⁷ 136  1.7 × 10⁻⁸ 143  9.4 × 10⁻⁷ 150  1.7 × 10⁻⁶

Schematic Representations for General Preparation of all Classes ofCompounds

Table 1

Compounds can be made by an adaption of the general route described byE. Schön et al., Biol. Chem. Hoppe-Seyler, 1991, 372, 305-311.

TABLE 2 (a) R: —CN

(b) R: —CH═NPh

(c) R:

For R¹ = —Ac

(R¹ = H) (d) R = —C≡CR

TABLE 3

TABLE 4 (W, P = Protecting groups; P¹, P² = Groups as described incorresponding tables) (a) R = CN

(IV) was prepared via method of G. Luisi et al., Tet. Lett., 1993, 34,2391-2392.

(c) For R=H, modify above procedure as described for Table 1 examples.

TABLE 5 (a) R = CN

(b) R=H, modify above procedure as described for Table 1 examples.

TABLE 6 Use method described for Table 5 examples for preparation of(VI) from (V)

TABLE 7 Standard coupling, dehydration and deprotection sequence similarto above schemes.

TABLE 8

Thioamides were prepared by the method described by K. Clausen et al.Tetrahedron, 1981, 37, 3635-3639. Other amide bioisosteres can beprepared from literature precedent (A. F. Spatola in “Chemistry andBiochemistry of Amino Acids, Peptides and Proteins”, Vol. III, B.Weinstein Ed., Marcel Dekker, New York, 1983, p. 267).

Experimental Details for Specific Examples

EXAMPLE 1 2-(S)-Cyano-1-isoleucylpyrrolidine (11)

Di-isopropylethylamine was added to a solution of H-ProNH₂. HCl (225 mg,1.50 mmol) in dry CH₂Cl₂ (15 cm³) until the pH was adjusted to 9.BocIleONSu was added in one portion and the mixture stirred for 16 h,under a nitrogen atmosphere. The solvent was evaporated and the residuetreated in the standard way, i.e. the residue was partitioned betweenethyl acetate (60 cm³) and 0.3 N KHSO₄ solution (10 cm³). The organiclayer was further washed with saturated NaCHO₃ solution (10 cm³), water(10 cm³) and brine (5 cm³). The solution was dried (Na₂SO₄) andevaporated at reduced pressure. The crude product was passed down ashort plug of silica gel, eluting with hexane:ethyl acetate, (10:90 to0:100) to yield 301 mg (92%) of BocIleProNH₂ as a colourless foam.

¹H NMR (CDCl₃), δ (ppm); 6.90 (1H, br.s); 5.51 (1H, br.s); 5.18 (1H, d,J=9.6 Hz); 4.62 (1H, dd, J=2.6, 7.0 Hz); 4.29 (1H, dd, J=8.4, 9.2 Hz);3.79-3.58 (2H, m); 2.36 (1H, m); 2.09-157 (5H, m); 1.43 (9H, s); 1.17(1H, m); 0.95 (3H, d, J=6.6 Hz); 0.90 (3H, t, J=7.3 Hz).

Imidazole (84 mg, 1.24 mmol) was added to a solution of BocIleProNH₂ indry pyridine (10 cm³), under a nitrogen atmosphere. The solution wascooled to −35° C., before the dropwise addition of POCl₃ (0.25 cm³, 2.48mmol). The reaction was stirred at −30° C. to −20° C. for 60 min. Thesolution was then evaporated and the crude residue subjected to columnchromatography (silica gel) to yield 180 mg (94%) of2-(S)-cyano-1-[N-(t-butoxycarbonyl) isoleucyl]pyrrolidine as acolourless oil.

¹H NMR (CDCl₃), δ (ppm); 5.14 (1H, d, J=92 Hz); 4.80 (1H, dd, J=26, 7.1Hz); 4.22 (1H, dd, J=7:9, 9.1 Hz); 3.81 (1H, m), 3.71 (1H, m), 2.30-2.12(4H, m); 1.75 (1H, m); 1.60 (1H, m); 1.42 (9H, s); 1.19 (1H, m); 0.97(3H, d, J=6.9 Hz); 0.91 (3H, t,J=7.3 Hz).

¹³C NMR (CDCl₃), δ (ppm); 171.7, 155.6, 118.0, 79.6, 56.0, 46.5, 46.0,37.8, 29.6, 28.1, 25.0, 24.2, 15.2, 10.9.

Deprotection was carried out by stirring with trifluoroacetic acid for60 min. Evaporation and lyophilisation from water afforded 60 mg of2-(S)cyano-1-isoleucylpyrlidine (11) as a white, fluffy solid.

FAB Mass Spec: Calculated 209.3, Found (M+H)⁺=210.2.

¹H NMR (D₂O), δ (ppm); 4.3 (1H, m); 3.64 (1H, d, J=5.6 Hz); 3.16 (2H,m); 1.86-1.48 (5H, m); 0.98 (1H, m); 0.68 (1H, m); 0.51 (3H, d, J=6.9Hz); 0.38 (3H, t, J=7.3 Hz).

¹³NMR (D₂O), δ (ppm); 169.7, 119.7, 57.3, 48.6, 48.1, 36.9, 30.2, 25.8,24.5, 15.4, 11.5.

EXAMPLE TWO H-Glu[NH(CH₂)₇CONH(CH₂)₃NHZ]pyrrolidide (64)

Di-isopropylethylamine was added to a solution of BocGlu(OH)pyrrolidide(193 mg, 0.64 mmol) and PyBop (500 mg, 0.96 mmol) in CH₂Cl₂ (6 cm³) toadjust the pH of the mixture to 9. After sting for 5 min, a solution ofbenzyl 8-amino otanoate (220 mg, 0.77 mmol) in CH₂Cl₂ (5 cm³) was added.The mixture was stirred at room temp for 16 h. The reaction was workedup in the standard procedure as described in example one. The cruderesidue was subjected to column chromatography (1% to 3% methanol inethyl acetate) to obtain 344 mg (99%) ofBocGlu[NH(CH₂)₇CO₂Bn]pyrrolidide as a colourless solid

¹H NMR (CDCl₃), δ (ppm); 7.35 (5H, s); 6.63 (1H, br.t, J=6.7 Hz); 5.65(1H, d, J=8.3 Hz); 5.11 (2H, s); 4.36 (1H, dt, J=2.6, 8.9 Hz); 3.55-3.20(6H, m); 2.34 (2H, t, J=7.3 Hz); 2.26 (2H, dd, J=5.6, 7.3 Hz); 2.11-1.48(10H, m); 1.43 (9H, s); 1.32-1.27 (6H, m).

Hydrogen gas was bubbled through a solution ofBocGlu[NH(CH₂)₇(CO₂Bn]pyrrolidide (230 mg, 0.43 mmol) in ethyl acetate(10 cm³), containing 10% palladium on charcoal (50 mg). After 90 min,the reaction vessel was flushed with nitrogen, the solution filteredthrough a pad of celite and the solvent evaporated to yield 187 mg (98%)of BocGlu[NH(CH₂)₇CO₂H]pyrrolidide as a colourless oil.

Di-isopropylethylamine was added to a solution ofBocGlu[NH(CH₂)₇CO₂H]pyrrolidide (125 mg, 0.28 mmol) and PyBop (221 mg,0.43 mmol) in CH₂Cl₂ (10 cm³) to adjust the pH of the solution to 9.After stirring for 5 min, a solution of ZNH(CH₂)₃NH₂. HCl (90 mg, 0.37mmol) and di-isopropylethylamine (38 mg, 0.37 mmol) was added in oneportion. The solution was stirred for 18 h then treated in the standardprocedure as described for example one. The crude residue was subjectedto column chromatography (2% to 15% methanol in ethyl acetate) to afford151 mg (85%) of BocGlu[NH(CH₂ ₇CONH(CH₂)₃NHZ]pyrrolidide as a colourlessoil.

¹H NMR (CDCl₃), δ (ppm); 7.35 (5H, s); 6.60 (1H, br.t, J=7.2 Hz); 6.14(1H, br.t, J=7.2 Hz); 5.63 (1H, d, J=8.3 Hz); 5.39 (1H, br.t, J=5.6 Hz);5.10 (2H, s); 4.38 (1H, dt, J=2.3, 9.2 Hz); 3.52-3.13 (1OH, m); 2.26(2H, t, J=6.9 Hz); 2.17 (2H, t, J=7.6 Hz); 1.98-1.48 (12H, m); 1.44 (9H,s); 1.38-123 (6H, m).

A solution of BocGlu[NH(CH₂)₇CONH(CH₂)₃NHZ]pyrrolidide (14 mg, 0.022mmol) in 4N HCl/dioxan was stirred for 45 min. The solvent wasevaporated and the residue dissolved in water, filtered and lyophilisedto yield 13 mg of H-Glu[NH(CH₂)₇CONH(CH₂)₃NHZ]pyrrolidide (64) as acolourless oil.

FAB Mass Spec: Calculated 531.3, Found (M+H)+=532.3.

EXAMPLE THREE H-Lys[CO(CH₂)NHSO₂Pfp]pyrrolidide (110)

ZNH(CH₂)₃CO₂NSu (570 mg, 1.7 mmol) was added in one portion to asolution of 1-[N-(t-butoxycarbonyl)lysyl]pyrrolidine (745 mg, 2.2 mmol)in dry CH₂Ca₂. The pH was adjusted to 9 with di-isopropylethylamine andthe mixture stirred for 60 min. The solvent was evaporated and theresidue treated in the standard procedure as described for example one.Column chromatography (100% ethyl acetate to 15% methanol in ethylacetate) afforded 620 mg (68%) of BocLys[CO(CH₂)₃NHZ]pyrrolidide.

¹H NMR (CDCl₃), δ (ppm); 7.42 (5H, s); 6.31 (1H, br.t, J=6.5 Hz); 5.58(1H, d, J=8.9 Hz); 5.39 (1H, br.t, J=6.9 Hz); 5.17 (2H, s); 4.44 (1H,m); 3.72-3.20 (8H, m); 2.29 (2H, t, J=7.3 Hz); 2.14-1.83 (8H, m);1.78-1.41 (4H, m); 1.43 (9H, s).

Hydrogen gas was bubbled through a mixture ofBocLys[CO(CH₂)₃NHZ]pyrrolidide (620 mg, 1.16 mmol) and 10% palladium oncharcoal in methanol (10 cm³) containing one molecular equivalent of 2NHCl. After 60 min, the reaction was flushed with nitrogen, and filteredthomugh celite. Evaporation of the solvent afforded 282 mg (49%) ofBocLys[CO(CH₂)₃NH₂. HCl]pyrrolidide. This product was dissolved inCH₂Cl₂ (10 cm³) and stirred, under a nitrogen atmosphere.Di-isopropylethylamine was added to adjust the pH to 9 before theintroduction of pentafluorobenzenesulfonyl chloride (45 mg, 0.17 mmol).This mixture was stirred for 16 h. The solvent was evaporated and thecrude material treated in the standard procedure described in exampleone. Column chromatography (100% ethyl acetate to 10% methanol in ethylacetate) afforded 33 mg (31%) of BocLys[CO(CH₂)₃NHSO₂Pfp]pyrrolidide asa colourless oil.

¹H NMR (CDCl₃), δ (ppm); 7.19 (1H, br.t, J=6.3 Hz); 6.18 (1H, br.t,J=6.6 Hz); 5.50 (1 d, J=8.4 Hz); 4.38 (1H, m); 3.65-3.16 (8H, m); 2.36(2, t, J=6.8 Hz); 2.01-1.82 (8H, m); 1.69-1.41 (4H, m); 1.43 (9H, s).

This product was stirred in trifluoroacetic acid (10 cm³) for 30 min.The solvent was evaporated and the residue dissolved in water, filteredand lyophilised to yield 30 mg of H-Lys[CO(CH₂)₃NHSO₂Pfp]Pr1 (110) as acolourless oil.

FAB Mass Spec: Calculated 514.2; Found (M+H)⁺=515.2.

EXAMPLE FOUR H-Thr[(CH₂₎ ₅CH₃]pyrolidide (143)

Pyrrolidine (0.88 g, 12.4 mmol) was added to a solution of BocThrONSu(3.0 g, 9.5 mmol) in dry CH₂Cl₂ (30 cm³), under a nitrogen atmosphere.The reaction was stirred for 60 min at room temperature. The solvent wasevaporated and the residue was treated in the standard procedure asdescribed for example one. The residue was subjected to columnchromatography (hexane:ethyl acetate, 30:70) to afford 250 g (96%) of1-[N-(t-butoxycarbonyl)threonyl]pyrrolidine as a colourless oil.

¹H NMR (CDCl₃), δ (ppm); 5.52 (1H, d, J=6.5 Hz); 4.30 (1H, d, J=7.4 Hz);4.16 (2H, m); 3.72 (1H, m); 3.46 (3H, w); 1.98-1.82 (4H, m); 1.43 (9H,s); 1.19 (3H, d, J=7.1 Hz).

Sodium hydride (17 mg, 0.70 mmol) was added to a solution of1-[N-(t-butoxycarbonyl) threonyl]pyrrolidine in dry THF, at 0° C., undera nitrogen atmosphere. The mixture was stirred at 0° C. for 15 minbefore the introduction of n-hexyl iodide (200 mg, 0.94 mmol). Thereaction was then allowed to stir at room temperature for 16 h. Thesolvent was evaporated and the residue treated in the standard manner asdescribed in example one. The crude product was subjected to columnchromatography (hexane:ethyl acetate, 40:60) to afford 25 mg (10%) ofBocThr[(CH₂)₅CH₃]pyrrolidide (143).

¹H NMR (CDCl₃), δ (ppm); 5.50 (1H, d, J=6.9 Hz); 4.48 (1Hz m); 3.70-3.32(7H, m); 1.92-1.80 (6H, m); 1.52 (2H, m); 1.42 (9H, s); 1.30 (6H, m);1.22 (8H, d, J=6.9 Hz); 0.83 (3H, t, J=7.9 Hz).

BocThr[(CH₂)₅CH₃]pyrrolidide (20 mg, 0.06 mmol) was stirred in 4NHCl/dioxan (5 cm³) for 60 min. The solvent was evaporated, the residuetaken up in water, filtered and lyophilised to yieldH-Thr[(CH₂)₅CH₃]pyrrolidide (20 mg) as an orange oil. The product waspurified by reverse phase HPLC to afford 15 mg of (143) as a colourlessoil.

FAB Mass Spec: Calculated 256.2, Found (M+H)⁺=257.3.

EXAMPLE FIVE H-Ile-Ψ[CH═CH]Pyrrolidide (149)

1.6 N ^(n)Butyl lithium (0.50 cm³, 0.76 mmol) was added to a stirredsolution of cyclopentyl triphenyphosphonium bromide (287 mg, 0.69 mmol)in dry THF (6 cm³), under a nitrogen atmosphere, maintaining thetemperature at −30° C. After stirring for 60 min, the solution wasfurther cooled to −50° C. subsequent to the dropwise addition of asolution of N-(t-butoxycarbonyl)-L-isoleucinal (125 mg, 0.58 mmol,prepared by the method of Fehrentz and Castro, Synthesis, 1983, 676), indry THF (4 cm³). After the final addition, the reaction was allowed toslowly attain room temperature, over 3.5 h.

The reaction was quenched with saturated ammonium chloride solution (2cm³). This was diluted with water (10 cm³) and extracted with diethylether (3×20 cm³). The combined ethereal layers were washed with water(10 cm³), dried (Na₂SO₄) and evaporated to yield 187 mg (>100%) of crudeproduct. Column chromatography (90:10, hexane:Et₂O) afforded 53 mg (34%)of Boc-Ile-Ψ[CH═CH]pyirolidide as a colourless oil.

¹H NMR (CDCl₃), 8 (ppm); 0.84 (3H, t, J=6.9 Hz); 0.91 (3H, d, J=7.3 Hz);1.08 (1H, m); 1.44 (9H, s); 1.48 (1H, m); 1.64 (5H, m); 2.24-2.45 (4H,m); 4.08 (1H, br.s); 4.41 (1H, br.s); 5.12 (1H, dt, J=2.3, 8.9 Hz).

¹³C NMR(CDCl₃) δ (ppm); 155.8, 147.4, 119.1, 79.2, 54.8, 40.1, 34.2,29.6, 28.9, 26.8, 26.6, 26.1, 15.0, 12.1.

Treatment of this product with 4N HCl/dioxan for 35 min removed theBoc-protecting group. The reaction was evaporated, the residue dissolvedin water, filtered and lyophilised to yield 24 mg (63%) ofH-Ile-Ψ[CH═CH]pyrrolidide (149) as a foamy solid.

FAB Mass Spec: Calculated 167.2, Found (M+H)⁺=168.2.

EXAMPLES SIX AND SEVEN H-Ile[(2R)-cyano-Ψ(CH═CH)pyrrolidide] (150)H-Ile[(2S)-cyano-Ψ(CH═CH)pyrrolidide] (151)

N-(t-Butoxycarbonyl)-L-isoleucinal (2.40 g, 11.2 mmol) and2-oxy-1-triphenylphosphoranecyclopentane (4.61 g, 13.4 mmol, prepared bymethod of H. O. House and H. Babed, J. Org. Chem., 1963, 28, 90) wereheated, at reflux, in toluene, under a nitrogen atmosphere. After 15 h,the mixture was cooled, and the solvent evaporated. Columnchromatography (80:20, hexane:ethyl acetate) of the crude residueafforded 2.33 g (74%) of BocIle-Ψ[CH═CH]pyrrolidin-2-one as a colourlessoil.

¹H NMR (CDCl₃), δ (ppm); 6.29 (1H, dt, J=2.6, 9.2 Hz); 4.59 (1H, br.d);4.17 (1H, m), 2.82 (1H, m); 2.66-2.50 (2H, m); 2.34 (2H, t, J=7.8 Hz);1.96 (2H, q, J=7.6 Hz); 1.44 (1H, m); 1.43 (9H, s); 1.12 (1H, m), 0.89(3H, d, J=5.3 Hz); 0.88 (3H, t, J=6.9 Hz).

Diethylcyanophosponoacetate (0.30 cm³, 1.92 mmol) was added to asolution of BocIle-Ψ[CH═CH]pyrrolidin-2-one (180 mg, 0.64 mmol) and LiCN(0.5 M in DMF, 3.84 cm³, 1.92 mmol) in dry DMF (2 cm³), under a nitrogenatmosphere. The reaction was stirred at room temperature for 30 min. Themixture was diluted with water (20 cm³) and then extracted with ethylacetate (2×30 cm³). The combined organic layers were washed with water(5×10 cm³), dried (Na₂SO₄) and evaporated to afford 360 mg (>100%) ofcrude product A portion of this crude cyano-phosphonate (284 mg, 0.64mmol) was dissolved in dry THF, and stirred under nitrogen. tert-Butanol(47 mg, 0.64 mmol) was added, followed by the dropwise addition of asolution of samarium (II) iodide (0.1 M in THF, 192 cm³, 1.92 mmol).After the final addition, the reaction was sted for a further 30 minbefore the addition of 2N HCl (20 cm³). The mixture was extracted withdiethyl ether (3×30 cm³). The combined ethereal layers were washed with10% Na₂S₂O₃ solution (10 cm³), water (2×10 cm³) and brine (2×10 cm³).The solution was dried (Na₂SO₄), evaporated and the crude residuesubjected to column chromatography (90:10, hexane:ethyl acetate) toyield 122 mg (66%) of a diastereomeric mixture ofBocIle[2-(RS)-cyano-Ψ(CH═CH)pyrrolidine] as a colourless oil.

¹H NMR (CDCl₃), δ (ppm); 5.52 (1H, d, J=9.6 Hz); 4.5 (1H, br.s); 4.12(1H, m); 3.35 (1H, m); 2.57 (1H, m); 2.38 (1H, m); 2.17 (1H, m); 1.91(2, m); 1.69 (2H, m); 1.53 (1H, m); 1.43 (9H, s); 1.12 (1H, m); 0.92(1.5 H, d, J=7.3 Hz); 0.91 (1.5 H, d, J=7.3 Hz); 0.89 (15H,d,J=6.6 Hz);0.86 (1.5H,t,J=6.9 Hz).

Treatment of this diastereomeric mixture with 4N HCl/dioxan for 60 minremoved the protecting group. Evaporation of the solvent and subsequentreverse phase HPLC of the residue afforded the two pure diastereomers.

(150), (47 mg, 60%) FAB Mass Spec: Calculated 1922, Found (M+H)⁺=193.2.

(151), (28 mg, 36%) FAB Mass Spec: Calculated 192.2, Found (M+H)⁺=193.2.

Preparative methods described herein in relation to Tables 1-8 and inexamples one to seven form part of the present invention.

Abbreviations Boc tert-Butyloxycarbonyl Bn Benzyl BSA Bovine serumalbumin ^(n)Bu n-Butyl Ch Cyclohexyl DMF Dimethylformamide DMPDess-Martin Periodane EDTA Ethylenediaminetetraacetic acid FAB Fast atombombardment Gua Guanidinyl HPLC High performance liquid chromatography^(m)Hx n-Hexyl Mass Spec Mass spectrometry mCPBA meta-Chloroperbenzoicacid Mol Wt Molecular weight OMSu N-O-Succinimide Pfp PentafluorophenylPh Phenyl Pip Piperidyl Prl Pyrrolidide Py Pyridine PyBopBenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphateWSCD Water soluble carbodiimide Z Benzyloxycarbonyl

What is claimed is:
 1. An inhibitor of DP-IV mediated processes ofgeneral formula:

n=1 or 2; m=0, 1, or 2; X=CH₂, O, S, SO, SO₂, NH or NR₁, where R₁=C₁-C₆alkyl; —Y=—N, —CH, or ═C, provided that when Y is ═C, the carbonyl groupof A is replaced by —CH═ or —CF═; R=H, CN, CHO, B(OH)₂, C≡C—R₇ orCH═N—R₈, where R₇=H, F, C₁-C₆ alkyl, CN, NO₂, OR₉, CO₂R₉ or COR₉;R₉=C₁-C₆ alkyl; R₈=Ph, OH, OR₉, OCOR₉ or OBn; A is attached to Y; and Ais

when R is H, CN, C≡C—R₇ or CH═N—R₈ where a=1-5;D=—G—(CH₂)_(b)—(R₄)_(q)—R₃; G=O, NH, NMe; b=0-12; q=0-5; D¹=D with G≠O;R₄=Z—NH—(CH₂)_(c)— or NH—Z—(CH₂)_(c)— where c=1-12 and Z=CO, CH₂ or SO₂;R₃=CO₂H or ester thereof, CONH₂, CONHNH₂, CONR₅R₆, CONHNR₅R₆, PO₃H orester thereof, SO₃H, SO₂NH₂, SO₂NR₅R₆, OH, OR₅, substituted orunsubstituted aryl or heteroaryl, NH₂, NR₅R₆, NHCO₂R₅, NHSO₂NR₅R₆,—NHCOR₅, NH—SO₂R₅, NH—CH(:NR₅R₆), NHCONR₅R₆, sugar, CO-aminosugar,NHCO-aminosugar or NHCS-aminosugar; and R₅ and R₆ are independendyselected from H and lower alkyl, fluoroalkyl, and cycloalkyl groups ofup to 8 atoms and aryl, heteroaryl and alkyl heteroaryl groups of up to11 atoms or R₅ and R₆ may together comprise a chain (C₃ to C₈); or A is

wherein R¹=H or Me, the ring may contain more heteroatoms,E=J—(CH₂)_(b)—(R₄)_(q)—R₃, J=CO, CH₂ or SO₂, and a, b, q, R₃ and R₄ areas defined under (i); or A is

wherein R₂=H or Me, the ring may contain one or more heteroatoms, andL=(CH₂)_(d)—(CO)—(CH₂)_(b)—(R₄)_(q)—R₃ or(CH₂)_(c)—NR₁—(CH₂)_(b)—(R₄)_(q)—R₃ where r=0 or 1, d=0-4, e=2-4, and b,q, R₃ and R₄ are as defined under (i); and wherein at least one CH₂group in a side chain may be replaced by a bioisostere thereof or anyamide group which connects A and B or which is in a side-chain of A maybe placed by an amide bioisostere.
 2. An inhibitor of a DP-IV mediatedprocess according to claim 1 selected from the group consisting of:N-(N^(ω)-(Benzyloxycarbonylmethyl)asparaginyl)pyrrolidine,N-(N^(ω)-(Carboxymethyl)asparaginyl)pyrrolidine,N-(N^(ω)-(3-Carboxypropyl)asparaginyl)pyrrolidine,N-(N^(ω)-(2-(Benzyloxycarbonyl)ethyl)asparaginyl)pyrrolidine,N-(N^(ω)-(2-Carboxyethyl)asparaginyl)pyrrolidine,N-(N^(ω)-(5-(Benzyloxycarbonyl)pentyl)asparaginyl)pyrrolidine,N-(N^(ω)-(5-Carboxypentyl)asparaginyl)pyrrolidine,N-(N^(ω)-(3-(Benzyloxycarbonyl)propyl)asparaginyl)pyrrolidine,N-(N^(ω)-(Benzyloxycarbonylmethyl)glutaminyl)pyrrolidine,N-(N^(ω)-(Carboxymethyl)glutaminyl)pyrrolidine,N-(N^(ω)-(2-(Benzyloxycarbonyl)ethyl)glutaminyl)pyrrolidine,N-(N^(ω)-(3-(Benzyloxycarbonyl)propyl)glutaminyl)pyrrolidine,N-(N^(ω)-(3-Carboxypropyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Benzyloxycarbonyl)pentyl)glutamiyl)pyrrolidine,N-(N^(ω)-(5-Carboxypentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(2-Carboxyethyl)glutaminyl)pyrrolidine,N-(N^(ω)-(7-(Benzyloxycarbonyl)heptyl)glutinyl)pyrrolidine,N-(N^(ω)-(7-Carboxyheptyl)glutaminyl)pyrrolidine,N-(N^(ω)-(7-(3-(Benzyloxycarbonylamino)propylarninocarbonyl)heptyl)glutamninyl)pyrrolidine,N-(N^(ω)-(6-(5-(Benzyloxycarbonyl)penylamocarbonyl)hexyl)glutaminyl)pyrrolidine,N-(N^(ω)-(6-(5-Carboxypentylaniiocarbonyl)hexyl)glutaminyl)pyrrolidine,N-(N^(ω)-(7-(3-Aminopropylaminocarbonyl)heptyl)glutaminyl)pyrrolidine,N-(N^(ω)-(11-(Benzyloxycarbonyl)undecyl)glutaminyl)pyrrolidine,N-(N^(ω)-(11-Carboxyundecyl)glutaminyl)pyrrolidine,N-(N^(ω)-(6-(Benzyloxycarbonyl)hexyl)glutaminyl)pyrrolidine,N-(N^(ω)-(6-Carboxyhexyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(2,2,2-Trifluoroethylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(2,2,3,3,4,4,4-Heptafluorobutylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(6-Hydroxyhexylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(3-Phenylpropylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(4-Phenylbutylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Dibutylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Dihexylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Benzylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(4-(Benzyloxycarbonyl)butyl)glutaminyl)pyrrolidine,N-(N^(ω)-(4-Carboxybutyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Ethylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(6-Hydroxyhexyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Piperidine-1-carbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-Carbamoylpentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Decylaninocarbonyl)pentyl)glutaninyl)pyrrolidine,N-(N^(ω)-(5-(Heptylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Cyclohexylmethylaniinocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(3-(Benzyloxycarbonylamino)propylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(3-Aminopropyannocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(3-Guanidinopropylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(4-Sulfoxyphenylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(1-Benzylpiperidin-4-ylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Piperidin-4-ylaminocarbonyl)pentyl)glutaminyl)pyrrolidine,N-(N^(ω)-(4-(N-Benkvloxycarbonyl-N-(3-benzyloxycarbonylaminopropyl)-aminocarbonyl)butyl)glutaminyl)pyrrolidine,N-(N^(ω)-(4-(3-Amiiopropylaminocarbonyl)butyl)glutaminyl)pyrrolidine,N-(N^(ω)-(5-(Benzyloxycarbonyl)pentyl)glutaminyl)prolinenitrile,N-(N^(ω)-(6-(5-(Benzyloxycarbonyl)pentylamninocarbonyl)hexyl)homoglutaminyl)-pyrrolidine,N-(N^(ω)-(6-(5-Carboxypentylaminocarbonyl)hexyl)homoglutaminyl)pyrrolidine,N-(N^(ω)-(5-(Benzyloxycarbonyl)pentyl)homoglutaminyl)pyrrolidine,N-(N^(ω)-(5-Carboxypentyl)homoglutaminyl)pyrrolidine,(3S)-3-Amino-N-(5-carboxypentyl)4-oxo4-(1-pyrrolidino)butanesulfonamide,N-(N^(ω)-(8-(Glucosaminothiocarbonylamino)octyl)glutninyl)pyrrolidine,N-((2S)-2-Amino-3-(7-carboxyheptanoylamino)propanoyl)pyrrolidine,N-((2S)-2-Amino-3-(7-(benzyloxycarbonyl)heptanoylanino)propanoyl)pyrrolidine,N-(N^(ω)-(5-Carboxypentanoyl)ornithinyl)pyrrolidine,N-(N^(ω)-(5-(Methyloxycarbonyl)pentanoyl)ornithinyl)pyrrolidine,N-(N^(ω)-(6-Aminohexanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-Aminobutanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-(Pentafluorobenzenesulfonylamino)butanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-(Pentafluorobenzoylamino)butanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-(2,2,2-Trifluoroethanesulfonylamino)butanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(12-(7-(Benzyloxycarbonylamino)heptanoylamino)dodecanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(12-(7-Aminoheptanoylamino)dodecanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(6-(6-(6-(Benzyloxycarbonylamino(hexanoylamino)hexanoylamino)hexanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(6-(6-(6-Aminohexanoylamino)hexanoylamino)hexanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-Carboxybutanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-(Benzyloxycarbonyl)butanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(7-Aminoheptanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(8-Aminooctanoyl)lysinyl)pyrrolidine,N-(N^(ω)-Octadecanoyllysinyl)pyrrolidine,N-(N^(ω)-(7-Guanidinoheptanoyl)lysinyl)pyrrolidine,N-(N^(ω)-Octanesulfonyllysinyl)pyrroiidine,N-(N^(ω)-(12-Aminododecanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(2-(Benzyloxycarbonylamino)ethanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(3-(Benzyloxycarbonylamino)propanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4(Benzyloxycarbonylamino)butanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(3-Aminopropanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(6-(Benzyloxycarbonylamino)hexanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(2-Guanidinoethanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(3-Aminopropanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(3-Guanidinopropanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(4-Guanidinobutanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(6-Guanidinohexanoyl)lysinyl)pyrrolidine,N-(N^(ω)-(7-Aminoheptanoyl)lysinyl)prolinenitrile,N-(N^(ω)-(8Aminooctanoyl)lysinyl)prolinenitrile,N-(O-(2-(5-Carboxypentylamino)-2-oxoethyl)serinyl)pyrrolidine,N-(O-(2-(5-(Benzyloxycarbonyl)pentylamino)-2-oxoethyl)serinyl)pyrrolidine,N-(O-(2-(4-(Benzyloxycarbonyl)butylamino)-2-oxoethyl)serinyl)pyrrolidine,N-(O-(2-(4-Carboxybutylaiino)-2-oxoetbyl)serinyl)pyrrolidine,N-(O-Methylthreoninyl)pyrrolidine, N-(O-Ethylthreoninyl)pyrrolidine,N-(O-Hexylthreoninyl)pyrrolidine,N-(O-(2-(5-(Benzyloxycarbonyl)pentylamino)-2-oxoethyl)threoninyl)pyrrolidine,N-(O-(2-(5-Carboxypentylamino)-2-oxoethyl)threoninyl)pyrrolidine,N-(O-(2-(4(Benzyloxycarbonyl)butylamino)-2-oxoethyl)threoninyl)pyrrolidine,and N-(O-(2-(4-Carboxybutylamino)-2-oxoethyl)threoninyl)pyrrolidine. 3.A pharmaceutical composition comprising a DP-IV inhibiting amount of acompound according to claim 1 and a pharmaceutically acceptable carrier.4. A method of inhibiting DP-IV in a patient, which comprisesadministering to the patient an amount of a compound according to claim1 which is effective to inhibit DP-IV.